Chromatin immunoprecipitation on microarray analysis of Smad2/3 binding sites reveals roles of ETS1 and TFAP2A in transforming growth factor beta signaling.
The Smad2 and Smad3 (Smad2/3) proteins are principally involved in the transmission of transforming growth factor beta (TGF-beta) signaling from the plasma membrane to the nucleus. Many transcription factors have been shown to cooperate with the Smad2/3 proteins in regulating the transcription of target genes, enabling appropriate gene expression by cells. Here we identified 1,787 Smad2/3 binding sites in the promoter regions of over 25,500 genes by chromatin immunoprecipitation on microarray in HaCaT keratinocytes. Binding elements for the v-ets erythroblastosis virus E26 oncogene homolog (ETS) and transcription factor AP-2 (TFAP2) were significantly enriched in Smad2/3 binding sites, and knockdown of either ETS1 or TFAP2A resulted in overall alteration of TGF-beta-induced transcription, suggesting general roles for ETS1 and TFAP2A in the transcription induced by TGF-beta-Smad pathways. We identified novel Smad binding sites in the CDKN1A gene where Smad2/3 binding was regulated by ETS1 and TFAP2A. Moreover, we showed that small interfering RNAs for ETS1 and TFAP2A affected TGF-beta-induced cytostasis. We also analyzed Smad2- or Smad3-specific target genes regulated by TGF-beta and found that their specificity did not appear to be solely determined by the amounts of the Smad2/3 proteins bound to the promoters. These findings reveal novel regulatory mechanisms of Smad2/3-induced transcription and provide an essential resource for understanding their roles.
Koinuma
Daizo
D
Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research, Koto-ku, Tokyo 135-8550, Japan.
Tsutsumi
Shuichi
S
Kamimura
Naoko
N
Taniguchi
Hirokazu
H
Miyazawa
Keiji
K
Sunamura
Makoto
M
Imamura
Takeshi
T
Miyazono
Kohei
K
Aburatani
Hiroyuki
H
D001665:Binding Sites; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D006801:Humans; D007438:Introns / Q000235:genetics; D046228:Microarray Analysis; D011401:Promoter Regions, Genetic; D050784:Proto-Oncogene Protein c-ets-1 / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051899:Smad2 Protein / Q000378:metabolism*; D051900:Smad3 Protein / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D050656:Transcription Factor AP-2 / Q000378:metabolism*; D024363:Transcription Initiation Site; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; C494041:ETS1 protein, human; D050784:Proto-Oncogene Protein c-ets-1; D016755:Proto-Oncogene Proteins c-jun; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; C493673:TFAP2A protein, human; D018808:Transcription Factor AP-1; D050656:Transcription Factor AP-2; D016212:Transforming Growth Factor beta